Diabetes Volume 64, February 2015
485
Peter H. Albers,1,2 Andreas J.T. Pedersen,3 Jesper B. Birk,1 Dorte E. Kristensen,1 Birgitte F. Vind,3
Otto Baba,4 Jane Nøhr,2 Kurt Højlund,3 and Jørgen F.P. Wojtaszewski1
Human Muscle Fiber Type–
Specific Insulin Signaling:
Impact of Obesity and Type 2
Diabetes
Diabetes 2015;64:485–497 | DOI: 10.2337/db14-0590
Skeletal muscle is important for whole-body insulinstimulated glucose disposal (1), and skeletal muscle insulin
1Section of Molecular Physiology, Department of Nutrition, Exercise and Sports,
August Krogh Centre, University of Copenhagen, Copenhagen, Denmark
2Diabetes Research Unit, Novo Nordisk A/S, Maaloev, Denmark
3Department of Endocrinology, Diabetes Research Center, Odense University
Hospital, Odense, Denmark
4Section of Biology, Department of Oral Function and Molecular Biology, School of
Dentistry, Ohu University, Koriyama, Japan
Corresponding author: Jørgen F.P. Wojtaszewski, [email protected].
resistance is a common phenotype of obesity and type 2
diabetes (T2D) (2). Skeletal muscle is a heterogeneous tissue composed of different fiber types, which can be divided
according to myosin heavy chain (MHC) isoform expression. Studies in rodents show that insulin-stimulated glucose uptake in the oxidative type I fiber–dominant muscles
is higher than in muscles with a high degree of glycolytic
type II fibers (3–6). Whether this phenomenon is due to
differences in locomotor activity of individual muscles or
a direct consequence of the fiber-type composition is largely
unknown. In incubated rat muscle, insulin-induced glucose
uptake was higher (;100%) in type IIa (oxidative/glycolytic)
compared with IIx and IIb (glycolytic) fibers (7,8), suggesting that insulin-mediated glucose uptake is related to the
oxidative capacity of the muscle fiber. In humans, a positive correlation between proportions of type I fibers
in muscle and whole-body insulin sensitivity has been
demonstrated (9–11). Furthermore, insulin-stimulated
glucose transport in human muscle strips was associated
with the relative type I fiber content (12). Thus, it is likely
that human type I fibers are more important than type II
fibers for maintaining glucose homeostasis in response to
insulin. Indeed, a decreased proportion of type I fibers has
been found in various insulin resistant states such as the
metabolic syndrome (9), obesity (13,14), T2D in some
(10,13,14) but not all (12,15) studies and following bedrest (16), as well as in tetraplegic patients (17), and subjects with an insulin receptor gene mutation (18).
Received 10 April 2014 and accepted 26 August 2014.
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0590/-/DC1.
© 2015 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
SIGNAL TRANSDUCTION
Skeletal muscle is a heterogeneous tissue composed
of different fiber types. Studies suggest that insulinmediated glucose metabolism is different between
muscle fiber types. We hypothesized that differences
are due to fiber type–specific expression/regulation of
insulin signaling elements and/or metabolic enzymes.
Pools of type I and II fibers were prepared from biopsies
of the vastus lateralis muscles from lean, obese, and type 2
diabetic subjects before and after a hyperinsulinemiceuglycemic clamp. Type I fibers compared with type II
fibers have higher protein levels of the insulin receptor,
GLUT4, hexokinase II, glycogen synthase (GS), and pyruvate dehydrogenase-E1a (PDH-E1a) and a lower protein
content of Akt2, TBC1 domain family member 4 (TBC1D4),
and TBC1D1. In type I fibers compared with type II
fibers, the phosphorylation response to insulin was similar (TBC1D4, TBC1D1, and GS) or decreased (Akt and
PDH-E1a). Phosphorylation responses to insulin adjusted for protein level were not different between fiber
types. Independently of fiber type, insulin signaling was
similar (TBC1D1, GS, and PDH-E1a) or decreased (Akt
and TBC1D4) in muscle from patients with type 2 diabetes compared with lean and obese subjects. We conclude that human type I muscle fibers compared with
type II fibers have a higher glucose-handling capacity
but a similar sensitivity for phosphoregulation by insulin.
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Muscle Fiber Types and Insulin Signaling
Mechanisms for a fiber type–dependent regulation of
glucose uptake could involve altered abundance/regulation
of insulin-signaling elements and/or metabolic enzymes. In
rats, insulin receptor content and Akt and GLUT4 protein
abundance are higher in type I compared with type II
fiber-dominated muscles (4,5,19–21). Furthermore, in
rats, Akt phosphorylation under insulin stimulation is
highest in type I compared with type II fiber-dominant
muscles (20). In humans, GLUT4 protein levels are higher
in type I compared with type IIa and IIx muscle fibers
(14,22). Overall, these findings suggest that insulin signaling to and effect on glucose transport is highest in type
I fibers. Thus, a shift toward reduced type I and hence
higher type II fiber content in obesity and T2D (10,13,14)
could negatively influence muscle insulin action on glucose metabolism. Insulin resistance in obesity and T2D is
characterized by a decreased ability of insulin to induce
signaling proteins proposed to mediate GLUT4 translocation by, for example, phosphorylation/activation of Akt
(23–25) and/or TBC1 domain family member 4 (TBC1D4)
(23,25). Whether this relates to differences in the response to insulin between fiber types is unknown.
Intracellular glucose metabolism could also be different
between muscle fiber types. Glucose entering the muscle
cell is initially phosphorylated by hexokinase (HK) and
predominantly stored as glycogen or oxidized in the
mitochondria through processes regulated by glycogen
synthase (GS) and the pyruvate dehydrogenase complex,
respectively. HKII content is higher in human soleus
muscle (;70% type I fibers) compared with gastrocnemius
and vastus lateralis muscle (;50% type I fibers) (26). Also,
the content of the pyruvate dehydrogenase (PDH) complex
subunit E1a (PDH-E1a) is decreased in muscle of proliferatoractivated receptor g-coactivator-1a knockout mice (27),
concomitant with a switch toward reduced type I fiber
abundance (28). Furthermore, mitochondrial density is
higher in human type I compared with type II fibers (29).
In contrast, no fiber type–specific expression pattern of GS
has been shown (30). Altogether, these observations suggest that glucose phosphorylation and oxidation but not
storage rate capacity are enhanced in type I compared
with type II fibers. Whether HKII and PDH-E1a abundance
as well as GS and PDH-E1a regulation by insulin is different
between human muscle fiber types is unknown.
We investigated whether proteins involved in glucose
metabolism were expressed and/or regulated by insulin in
a fiber type–specific manner in human skeletal muscle.
This was achieved by creating pools of single fibers
expressing either MHC I (type I) or II (type II). These
fibers were dissected from vastus lateralis muscle biopsies
obtained from lean and obese normal glucose-tolerant
subjects as well as T2D patients.
Diabetes Volume 64, February 2015
studies conducted at Odense University Hospital (Odense,
Denmark). One fraction (eight lean, seven obese, and six
T2D) was from an already published study (31), while the
remaining subjects were from an unpublished study, in
which subjects were investigated with an identical experimental protocol as previously described (31). Both studies were approved by the regional ethics committee and
carried out in accordance with the Declaration of Helsinki
II. Subject medication is detailed in the Supplementary
Material.
Experimental Protocol
A detailed explanation of the in vivo study protocol has
been published elsewhere (31). In short, all subjects were
instructed to refrain from strenuous physical activity 48 h
before the experimental day. After an overnight fast, subjects underwent a 2-h basal tracer equilibration period
followed by a 4-h hyperinsulinemic-euglycemic clamp
(Actrapid; Novo Nordisk) at an insulin infusion rate of
40 mU $ m22 $ min21 combined with tracer glucose and indirect calorimetry. A primed-constant [3-3H]glucose infusion
was used throughout the 6-h study, and [3-3H]glucose
was added to the glucose infusates to maintain plasmaspecific activity constant at baseline levels during the 4-h
clamp period as described in detail previously (32). Vastus
lateralis muscle biopsies were obtained before and after
the clamp under local anesthesia (1% lidocaine) using a
modified Bergström needle with suction. Muscle biopsies
were immediately frozen in liquid nitrogen and stored
below 280°C.
Dissection of Individual Muscle Fibers
Muscle fibers were prepared as previously described (33)
but with minor modifications. A total of 20–60 mg of
muscle tissue was freeze-dried for 48 h before dissection
of individual muscle fibers in a climate-controlled room
(20°C, ,35% humidity) using a dissection microscope (in
total, n = 5,384 fibers from 64 biopsies). The length of
each fiber was estimated under the microscope (1.5 6 0.4
mm [means 6 SD]) before being carefully placed in a PCR
tube and stored on dry ice. On the day of dissection, 5 mL
of ice-cooled Laemmli sample buffer (125 mmol/L TrisHCl [pH 6.8], 10% glycerol, 125 mmol/L SDS, 200 mmol/L
dithiothreitol, and 0.004% bromophenol blue) was added
to each tube. During method optimization, addition of protease and phosphatase inhibitors was found to be unnecessary for preservation of either protein content or protein
phosphorylation for this type of sample preparation (data
not shown). After thorough mixing at 4°C, each tube was
inspected under a microscope to confirm that the fiber was
properly dissolved (if not, the tube was discarded). Each
sample was then heated for 10 min at 70°C and stored
at 280°C.
Preparation of Pooled Muscle Fiber Samples
RESEARCH DESIGN AND METHODS
Subjects
A total of 10 lean healthy, 11 obese nondiabetic, and 11
obese T2D subjects were randomly chosen from two
A small fraction (1/5) of the solubilized fiber was used for
identification of MHC expression using Western blotting and
specific antibodies against MHC I or II (see IMMUNOBLOTTING).
Hybrid fibers (;5%) expressing more than one MHC
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isoform were discarded. Pools of type I and II fibers from
each biopsy were prepared (128 pools in total). The average number of type I and II fibers per muscle biopsy included in each pool was 20 (range 9–36) and 42 (range
22–147), respectively.
Estimation of Protein Content and Test of Purity
Protein content of the fiber-specific samples was estimated using 4–20% Mini-PROTEAN TGX stain-free gels
(Bio-Rad), which allowed for gel-protein imaging following ultraviolet activation on a ChemiDoc MP Imaging System (Bio-Rad). The intensity of visualized protein bands
(from 37–260 kDa) was compared with a standard curve
from three different pools of human muscle homogenates
with a known protein concentration (Supplementary Fig. 1).
After gel imaging, the purity of each pooled sample was
re-evaluated using Western blotting and MHC I– and II–
specific antibodies (see IMMUNOBLOTTING). All fiber-specific
samples were diluted with Laemmli sample buffer to a protein concentration of 0.2 mg/mL.
Glycogen Determination in Muscle Fiber Pools
Glycogen content in the fiber-specific pools was measured
by dot blotting using a specific antibody against glycogen
(34,35). Briefly, 150 ng of protein was spotted onto a polyvinylidene difluoride membrane. After air drying, the
membrane was reactivated in ethanol before blocking, incubation in primary and secondary antibody, and visualization as described in IMMUNOBLOTTING. The intensity of
each dot was compared with a standard curve (Supplementary Fig. 2) obtained from a muscle homogenate
with a glycogen content predetermined biochemically as
previously described (31) and expressed accordingly.
MHC Determination
For MHC determination in muscle biopsies, lysates were
prepared, and protein content was measured as previously
described (31). Muscle lysates were diluted 1:3 with 100%
glycerol/Laemmli sample buffer (50/50) and run on 8% selfcast stain-free gels containing 0.5% 2,2,2-trichloroethanol
(36). A total of 3 mg of lysate protein was separated
for ;16 h at 140 V as previously described (37). Protein
bands were visualized by ultraviolet activation of the stainfree gel on a ChemiDoc MP Imaging System (Bio-Rad) and
quantified as stated below. Coomassie staining of the gel
and the use of muscle homogenates provided similar results as stain-free gel imaging and muscle lysates, respectively (data not shown).
Immunoblotting
For MHC determination of single muscle fibers and
evaluation of total and phosphorylated levels of relevant
proteins, equal amounts of sample volume (for MHC
determination) or protein amount were separated using
either precast (Bio-Rad) or self-cast 7.5% gels. On each gel,
an internal control (muscle lysate) was loaded two times
per gel in order to minimize assay variation. Muscle fiber
pool values were divided by the average of the internal
control sample from the corresponding gel. Furthermore,
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487
on one gel, a standard curve of muscle homogenate was
loaded to ensure that quantification of each protein probed
for was within the linear range. Following separation,
proteins were transferred (semidry) from multiple gels to
a single polyvinylidene difluoride membrane that was
incubated with blocking agent (0.05% Tween 20 and 2%
skimmed milk in Tris-buffered saline) for 45 min at room
temperature, followed by incubation in primary antibody
solution overnight at 4°C (for antibody details, see Supplementary Table 1). Membranes were incubated with appropriate secondary antibodies (Jackson ImmunoResearch
Laboratories) that were conjugated to either horseradish
peroxidase or biotin for 1 h at room temperature. Membranes incubated with biotin-conjugated antibody were
further treated with horseradish peroxidase–conjugated
streptavidin. Protein bands were visualized using a ChemiDoc
MP imaging system (Bio-Rad) and enhanced chemiluminescence (SuperSignal West Femto; Pierce). Band
densitometry was performed using Image Laboratory (version 4.0). Membranes were reprobed with an alternate
antibody according to the scheme given in Supplementary
Table 2.
Statistical Analyses
Subject characteristics and blood parameters were evaluated by a one-way ANOVA. To compare fiber type, insulin,
and group effects, a three-way ANOVA with repeated
measures for fiber type and insulin was used. If no triple
interaction was present, a two-way ANOVA on the increment with insulin (Dinsulin-basal values) was performed
for fiber type and group effects with repeated measures for
fiber type. Main effects of group and significant interactions were evaluated by Tukey post hoc testing. Statistical
analyses were performed in SigmaPlot (version 12.5, Systat
Software; one- and two-way ANOVA) and in SAS statistical
software (version 9.2, SAS Institute; three-way ANOVA).
Unless otherwise stated, n equals number of subjects as
indicated in Table 1. Differences were considered significant at P , 0.05.
RESULTS
Clinical and Metabolic Characteristics
BMI and fat mass were higher in the obese and T2D groups
compared with the lean group (Table 1). Patients with T2D
compared with lean and obese subjects had elevated HbA1c
levels, increased fasting plasma glucose, insulin, and triglyceride (vs. lean only) concentrations (Tables 1 and 2). During the hyperinsulinemic-euglycemic clamp, the glucose
disposal rate (GDR) was decreased in T2D versus lean
and obese subjects (Table 2). The decrease in GDR resulted
from both lower glucose oxidation rates and reduced nonoxidative glucose metabolism (Table 2).
Fiber Type Composition
In muscle biopsies from lean and obese subjects, MHC I,
IIa, and IIx constituted 45, 46, and 9% (total 55% MHC
II), respectively (Fig. 1A). This fiber type composition
is in accordance with previous observations using
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Muscle Fiber Types and Insulin Signaling
Diabetes Volume 64, February 2015
Table 1—Subject characteristics at study entry
Lean
Obese
T2D
n (female/male)
10 (2/8)
11 (2/9)
11 (2/9)
Age (years)
54 6 2
56 6 2
55 6 2
Height (m)
1.77 6 0.03
1.77 6 0.03
1.75 6 0.03
BMI (kg/m2)
23.9 6 0.4
30.5 6 0.6***
30.8 6 1.0***
Fat-free mass (kg)
59.3 6 3.3
68.5 6 3.5
63.3 6 3.3
Fat mass (kg)
16.2 6 0.6
28.1 6 1.1***
31.8 6 2.7***
HbA1c (%)
5.4 6 0.1
5.2 6 0.1
6.8 6 0.2***,†††
35 6 1
34 6 1
51 6 3***,†††
HbA1c (mmol/mol)
Plasma cholesterol (mmol/L)
5.5 6 0.3
5.6 6 0.2
5.0 6 0.2
Plasma LDL cholesterol (mmol/L)
3.6 6 0.2
3.7 6 0.2
2.9 6 0.2†
Plasma HDL cholesterol (mmol/L)
1.6 6 0.1
1.4 6 0.1
1.0 6 0.1**,†
Plasma triglycerides (mmol/L)
0.9 6 0.1
1.4 6 0.2
2.6 6 0.6*
—
—
4.0 6 1.5
Diabetes duration (years)
Values are means 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 vs. lean group; †P , 0.05, †††P , 0.001 vs. obese group.
(immuno)histochemistry (9–11,13–15,26) and biochemical
methods (18,22). In the T2D group, MHC I, IIa, and IIx
constituted 35, 45, and 20% (total 65% MHC II), respectively. In the T2D group compared with the lean and obese
group, the relative number of type I muscle fibers was lower,
and the relative number of type IIx muscle fibers was higher.
MHC IIa expression was similar among all three groups.
Fig. 3). Higher protein levels of insulin receptor b (16%),
HKII (470%), GLUT4 (29%), and electron transport
chain complex II (35%) was found in type I versus II
fibers (Fig. 1C–F). No differences between groups were
observed except for a reduced (224%) insulin receptor
b level in the T2D compared with the lean and obese
groups (Fig. 1C–F).
Insulin Receptor, HKII, GLUT4, and Complex II
Akt, Mammalian Target of Rapamycin, and N-myc
Downstream-Regulated Gene 1
As represented in Fig. 1B, all fiber pools contained one MHC
isoform only. Actin was used as a reference protein, and actin
abundance was equal between fiber pools (Supplementary
Akt2 protein content was lower (227%) in type I versus II
fibers (Fig. 2C). In the three groups, the average increases
Table 2—Metabolic characteristics during hyperinsulinemic-euglycemic clamp
Lean
Obese
T2D
Plasma glucosebasal (mmol/L)
5.6 6 0.2
5.9 6 0.1
9.0 6 0.6***,†††
Plasma glucoseclamp (mmol/L)
5.5 6 0.1
5.3 6 0.2
5.5 6 0.1
Serum insulinbasal (pmol/L)
27 6 3
44 6 5
86 6 15***,†
Serum insulinclamp (pmol/L)
408 6 23
399 6 12
422 6 17
76 6 3
77 6 2
80 6 4
161 6 24***,†††
GDRbasal (mg/m2/min)
388 6 28
334 6 20
Glucose oxidationbasal (mg/m2/min)
50 6 8
47 6 4
46 6 7
Glucose oxidationclamp (mg/m2/min)
141 6 14
126 6 10
77 6 7***,††
2
GDRclamp (mg/m /min)
NOGMbasal (mg/m2/min)
26 6 8
30 6 4
34 6 9
NOGMclamp (mg/m2/min)
247 6 22
208 6 23
84 6 22***,††
Lipid oxidationbasal (mg/m2/min)
28 6 2
30 6 2
34 6 3
Lipid oxidationclamp (mg/m2/min)
21 6 5
463
19 6 4**,†
0.82 6 0.01
0.81 6 0.01
0.80 6 0.01
RERclamp
0.98 6 0.03
0.95 6 0.02
0.87 6 0.02**,†
Plasma lactatebasal (mmol/L)
0.78 6 0.09
0.80 6 0.07
1.06 6 0.11
Plasma lactateclamp (mmol/L)
1.36 6 0.08
1.18 6 0.08
0.93 6 0.06***
RERbasal
Values are means 6 SEM. NOGM, nonoxidative glucose metabolism; RER, respiratory exchange ratio. **P , 0.01, ***P , 0.001 vs. lean
group; †P , 0.05, ††P , 0.01, †††P , 0.001 vs. obese group.
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Figure 1—MHC composition and muscle fiber type–specific protein abundance in lean, obese, and T2D subjects. A: MHC composition
measured in whole muscle biopsies from lean, obese, and T2D subjects. B: The purity of each muscle fiber pool was checked by Western
blotting of MHC I and II. Representative blots of MHC I and II muscle fiber pools from three subjects are shown. In muscle fiber pools, the
protein content of the insulin receptor b (C), HKII (D), GLUT4 (E), and electron transport complex II (F) was evaluated by Western blotting.
Quantified values of each protein (C–F) are related to the content of actin protein, and the basal type I fiber value in the lean group is set to
100. Representative blots from three individuals are shown above each bar in A and C–F. White bars represent type I fibers (A) or type I fiber
pools (C–F), black bars type IIa fibers (A) or type II fiber pools (C–F), and gray bars IIx fibers (A). Data are means 6 SEM. Post hoc testing
was only performed when an interaction was evident. †P < 0.05; †††P < 0.001 vs. type I muscle fibers; ‡P < 0.05, ‡‡P < 0.01 main effect
of group compared with lean; (§)P = 0.06, §P < 0.05, §§P < 0.01 main effect of group compared with obese. AU, arbitrary units.
under insulin stimulation of phosphorylated (p-)AktThr308
and p-AktSer473 were 5.8- and 3.5-fold in type I fibers and
6.1- and 3.7-fold in type II fibers, respectively (Fig. 2A and
B). In lean and obese groups, levels of insulin-stimulated
p-AktThr308 were lower (225%) in type I versus II fibers.
In the T2D group, the insulin-stimulated p-AktThr308 and
p-AktSer473 were lower in both fiber types compared with
lean and obese groups. In response to insulin, phosphorylation of AktSer473/Akt2 but not AktThr308/Akt2 was fiber
type dependent, although the relative response to insulin
was similar between fiber types (Supplementary Fig. 4A
and B). In type I fibers, a higher protein level of mammalian target of rapamycin (mTOR) (20%) and its downstream target N-myc downstream-regulated gene (NDRG)
1 (68%) compared with type II fibers was evident (Fig. 3B
and D). Insulin had no effect on p-mTOR2481 but increased
p-NDRG1Thr346 only in type I fibers from obese (86%)
and T2D (100%) groups (Fig. 3A and C). No fiber-type
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Diabetes Volume 64, February 2015
differences were evident when p-NDRG1Thr346 was adjusted for NDRG1 protein abundance (Supplementary
Fig. 4C).
TBC1D1 and TBC1D4
TBC1D1 and TBC1D4 protein levels were lower (245%
and 216%) in type I versus II fibers, respectively (Fig. 4B
and G). Irrespective of fiber type, insulin stimulation increased p-TBC1D1Thr596 (36%) and p-TBC1D4 at all sites
investigated (Ser318 [122%], Ser588 [59%], Thr642 [103%],
and Ser704 [113%]) (Fig. 4A and C–F). Statistically significant main effects of fiber type were evident for the level of
phosphorylation of both TBC1D1 and TBC1D4. More specifically, p-TBC1D1Thr596 (262%), p-TBC1D4Ser318 (221%),
p-TBC1D4Ser588 (221%), p-TBC1D4Thr642 (224%), and
p-TBC1D4Ser704 (224%) were lower in type I compared
with type II fibers. No significant group differences in protein abundance or protein phosphorylation of TBC1D1 and
TBC1D4 were evident, although the response to insulin of
p-TBC1D4Ser588 tended (P = 0.07) to be group dependent.
Glycogen Content, GS Kinase 3, and GS
In the basal state, glycogen content was lower (229%) in
type I versus II fibers in the lean (P , 0.001), obese (P =
0.09), and T2D (P = 0.09) groups (Fig. 5A). Insulin induced no significant changes in glycogen content in either
of the fiber types. The protein levels of GS kinase (GSK)
3b were 14% less in type I versus type II, whereas GS
protein was 53% higher in type I compared with type II
fibers (Fig. 5C and F). In all three groups and in both fiber
types, insulin induced a similar change in phosphorylation
of GSK3bSer9 (62%), GS2+2a (236%) and GS3a+b (238%)
(Fig. 5B, D, and E). Phosphorylation of GSK3bSer9 was
lower (231%), whereas phosphorylation of GSsite2+2a
and GSsite3a+b was, respectively, 68 and 51% higher in
type I versus II fibers. No significant differences were
evident between individual groups in protein abundance
and protein phosphorylation of GSK3b and GS.
PDH
Figure 2—Akt in muscle fiber pools from lean, obese, and T2D
subjects. Muscle fiber type–specific regulation of Akt phosphorylation on site Thr308 (A) and Ser473 (B) and protein content of Akt2
(C) was evaluated by Western blotting. Two bands are apparent
for human Akt2 when insulin stimulated [both being Akt2 (31)].
Quantified values of each protein are related to the content of actin
protein, and the basal type I fiber value in the lean group is set to
100. Representative blots are shown above bars for each protein
probed for. White bars represent type I and black bars type II muscle fiber pools. Data are means 6 SEM. Post hoc testing was only
performed when an interaction was evident. ***P < 0.001 vs. basal
conditions; ††P < 0.01 vs. type I muscle fibers; ‡P < 0.05, ‡‡P <
0.01, ‡‡‡P < 0.001 vs. lean group; §P < 0.05, §§P < 0.01, §§§P <
0.001 vs. obese group. AU, arbitrary units.
PDH-E1a protein content was 34% higher in type I versus
II fibers (Fig. 6C). Basal levels of PDH-E1a site 1 phosphorylation were similar between fiber types in all three
groups (Fig. 6A). After insulin, the degree of phosphorylation was significantly lower in type II versus I fibers in
the obese and T2D groups only, indicating dephosphorylation by insulin in type II but not in type I fibers. In line,
PDH-E1a site 2 phosphorylation was decreased by insulin, and this effect was dependent on fiber type toward
a greater effect of insulin in type II versus I fibers (Fig. 6B).
Fiber-type differences were not evident when p-PDHsite1
and p-PDHsite2 was adjusted for PDH-E1a content (Supplementary Fig. 4D and E).
DISCUSSION
The current study is the first to evaluate changes in
signaling events in response to insulin in fiber type–specific
pools from human muscle. Based on our findings, we propose a model in which human type I fibers have a greater
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Figure 3—mTOR and NDRG1 in muscle fiber pools from lean, obese, and T2D subjects. Muscle fiber type–specific regulation of mTOR
phosphorylation on site Ser2481 (A) and NDRG1 phosphorylation on site Thr346 (C) as well as protein content of mTOR (B) and NDRG1
(D) were evaluated by Western blotting. Two bands are apparent for both p-NDRG1Thr346 and NDRG1 (both quantified). Quantified values of
each protein are related to the content of actin protein, and the basal type I fiber value in the lean group is set to 100. Representative blots
are shown above bars for each protein probed for. White bars represent type I and black bars type II muscle fiber pools. Data are means 6
SEM. Post hoc testing was only performed when an interaction was evident. *P < 0.05; ***P < 0.001 vs. basal conditions; †††P < 0.001 vs.
type I muscle fibers. AU, arbitrary units.
abundance of proteins to transport (29% GLUT4), phosphorylate (470% HKII), and oxidize (35% electron transport chain complex II and 34% PDH) glucose and to
synthesize glycogen (35% GS) compared with type II
fibers. These observations are supported by significant
positive correlations between the MHC I content in whole
muscle lysates and insulin-stimulated GDR (r = 0.53; P =
0.002), glucose oxidation rate (r = 0.52; P = 0.003), and
nonoxidative glucose metabolism (r = 0.44; P = 0.01)
(Supplementary Fig. 5). Interestingly, even though insulin
receptor content was higher (16%) in type I fibers, phosphoregulation of TBC1D1, TBC1D4, and GS by insulin
was similar between fiber types (all normalized to actin).
The apparent fiber-type differences in insulin-stimulated
phosphorylation of Akt, NDRG1, and PDH-E1a (when
related to actin) were eliminated when adjusted for Akt2,
NDRG1, and PDH-E1a protein abundance. These findings
suggest a similar sensitivity of type I and II muscle fibers
for regulation by insulin of the proteins investigated.
Insulin-stimulated GDR, glucose oxidation rates, and
nonoxidative glucose metabolism were decreased in T2D
compared with the lean and obese groups. This was
accompanied by lower insulin receptor content and
altered response to insulin of p-Akt308, p-Akt473,
p-TBC1D4Ser588 (P = 0.07), and p-NDRG1Thr346 in the
muscle fiber–specific pools from the T2D compared with
the lean and obese groups. In cells, NDRG1 phosphorylation has been suggested to be a readout of mTOR complex
(mTORC) 2 activities (38). mTORC2 is also a widely accepted upstream kinase for AktSer473 (39). Since the response to insulin of p-NDRG1Thr346/NDRG1 was similar
between groups, these data could imply a specific dysfunctional link between mTORC2 and p-AktSer473, as the latter
was decreased in response to insulin in both type I and II
fibers in T2D compared with the lean and obese groups.
In rat muscle, abundance and insulin-stimulated phosphorylation of Akt were higher (660 and 160–180%, respectively) in soleus muscle primarily containing type I
fibers, as opposed to epitrochlearis and extensor digitorum longus muscles primarily consisting of type II fibers
(20). In contrast, in human muscle, we report a decreased
Akt phosphorylation after insulin in type I versus II fibers,
due to higher Akt2 levels in type II fibers. Thus, findings
in rat muscles with a diverse fiber-type composition could
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Muscle Fiber Types and Insulin Signaling
Diabetes Volume 64, February 2015
Figure 4—TBC1D1 and TBC1D4 in muscle fiber pools from lean, obese, and T2D subjects. Muscle fiber–specific regulation of TBC1D1
phosphorylation at site Thr596 (A) and TBC1D4 phosphorylation on site Ser318 (C), Ser588 (D), Thr642 (E), and Ser704 (F) as well as protein
content of TBC1D1 (B) and TBC1D4 (G) were evaluated by Western blotting. Two bands are apparent for p-TBC1D1Thr596 and TBC1D1
[long and medium/short isoform of TBC1D1 protein (49)]. Quantified values of each protein are related to the content of actin protein, and
the basal type I fiber value in the lean group is set to 100. Representative blots are shown above bars for each protein probed for. White
bars represent type I and black bars type II muscle fiber pools. Data are means 6 SEM. AU, arbitrary units.
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493
Figure 5—Glycogen content, GSK3b, and GS in muscle fiber pools from lean, obese, and T2D subjects. A: Muscle fiber–specific glycogen
content measured by dot blotting. Muscle fiber–specific phosphorylation of GSK3b on site Ser9 (B) and GS phosphorylation on site 2+2a
(D) and 3a+b (E) as well as protein abundance of GSK3b (C) and GS (F) were evaluated by Western blotting. Quantified values of each
protein (B–F) are related to the content of actin protein, and the basal type I fiber value in the lean group is set to 100. Representative blots
are shown above bars for each protein probed for. White bars represent type I and black bars type II muscle fiber pools. Data are means 6
SEM. Post hoc testing was only performed when an interaction was evident. (†)P = 0.09, †††P < 0.001 vs. type I muscle fibers. AU,
arbitrary units.
simply result from differences in locomotor activity, although species-related differences cannot be excluded. For
instance, TBC1D4 and TBC1D1 protein abundance in the
current study are only modestly lower (216% and 245%)
in human type I versus II fibers. In mice, a high (.10-fold)
TBC1D4 and a low (,20%) TBC1D1 content are evident in
the type I fiber–abundant soleus compared with the
type II fiber–abundant extensor digitorum longus muscle
(40). In rats, no significant correlations between MHC isoform abundance in various muscles and either TBC1D1 or
TBC1D4 protein content were found (21). These findings
indicate that fiber-type differences in TBC1D4 and
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Muscle Fiber Types and Insulin Signaling
Figure 6—PDH-E1a in muscle fiber pools from lean, obese, and
T2D subjects. Muscle fiber type–specific regulation of PDH-E1a
phosphorylation on site 1 (A) and site 2 (B) as well as PDH-E1a
protein content (C) were evaluated by Western blotting. Phosphospecific PDH-E1a antibodies were directed against the phosphorylation of sites Ser293 (site 1) and Ser300 (site 2) on the human
PDH-E1a isoform. Due to sample limitations, protein levels of
PDH-E1a were evaluated in a subset of fiber pools, with the number
of samples indicated in each bar. Quantified values of each protein
are related to the content of actin protein, and the basal type I fiber
value in the lean group is set to 100. Representative blots are
shown above bars for each protein probed for. White bars represent
type I and black bars type II muscle fiber pools. Data are means 6
SEM. Post hoc testing was only performed when an interaction was
evident. †††P < 0.001 vs. type I muscle fibers. AU, arbitrary units.
Diabetes Volume 64, February 2015
TBC1D1 protein levels are highly dependent on the species investigated.
In the current study, no differences in the response to
insulin were observed between fiber types in phosphorylation of TBC1D4 and TBC1D1. We previously reported
a decreased response to insulin of p-TBC1D4Ser318 and
p-TBC1D4Ser588 in skeletal muscle from obese T2D subjects compared with weight-matched control subjects
(23). In the current study, insulin-induced (delta values
[insulin minus basal]) p-TBC1D4Ser588 was borderline (P =
0.07) group dependent. The average response to insulin
was 62, 96, and 19% in the lean, obese, and T2D groups,
respectively. It has been shown that exercise training normalizes defects in insulin action on TBC1D4 regulation in
T2D versus control subjects (23). Thus, in the current
study, the lack of significant defects in TBC1D4 regulation
by insulin in the T2D group compared with control groups
could be due to the physical fitness level of the groups
studied. We found that p-TBC1D1Thr596 was increased by
insulin in agreement with another study (41) and that the
relative increase was irrespective of fiber type and group.
We conclude that the relative response to insulin of Akt,
TBC1D4, and TBC1D1 is independent of fiber type, while
the absolute amount of phosphorylated protein is lower
in type I versus II fibers. Whether a higher total amount
of phosphorylated protein is important for the regulation of glucose uptake is unknown. To investigate the
impact of the present findings on glucose uptake in different human muscle fiber types, future studies need to
examine the membrane-bound fraction of GLUT4 in different fiber types or even measure single muscle fiber
glucose transport as performed in rat muscle (7).
Interestingly, Gaster et al. (14) previously reported
that GLUT4 abundance was significantly lower in type I
fibers only in muscle from T2D patients compared with
lean and obese control subjects. This was not evident in
the current study. However, we found a nonsignificantly
lower GLUT4 content of the same magnitude (10–20%) as
previously reported (14) in both type I and II fibers from
the T2D compared with the lean and obese groups. Also,
GLUT4 levels were generally higher in type I versus II
fibers. Thus, fewer type I fibers in the T2D compared
with the lean and obese groups possibly lowers the glucose uptake capacity in diabetic skeletal muscle. In support, HKII content was higher in type I compared with
type II fibers. The influence of HKII protein levels on
glucose uptake is controversial and has recently been estimated to control ;10% of human skeletal muscle glucose metabolism during insulin-stimulated conditions
(42). In the current study, fiber type–specific HKII levels
were not different between groups investigated. Thus, it
is likely that decreased HKII levels reported in muscles
from T2D subjects (43) are at least partly influenced by
a lower number of type I fibers in T2D versus control
subjects as also shown in the current study. Interestingly, in contrast to HKII, HKI protein abundance was
lower (219%) among the three groups in type I versus II
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fibers (Supplementary Fig. 6). This observation could indicate a different role of HK isoforms in type I and II
muscle fibers.
A close correlation between the insulin-stimulated
increase in nonoxidative glucose metabolism and GS
activity has been reported (44). In the current study,
insulin-stimulated nonoxidative glucose metabolism
was decreased in the T2D compared with the lean and
obese groups as shown by others (23,31,41,45). Thus,
we investigated the fiber type–specific regulation of GS
by insulin. We were unable to detect any differences in
the response to insulin between fiber types, although the
absolute amount of phosphorylated GS was highest in
type I fibers. Increased phosphorylation of GS in type I
fibers could be accounted for by a higher GS protein level
in type I versus II fibers. Previously, a similar GS content
in type I, IIa, and IIx fiber pools was reported in muscle
from young (23 years) subjects (30). Thus, the present
findings of a higher GS content in type I versus II fibers
in muscle from middle-aged (;55 years) subjects indicates an age-dependent fiber type–specific regulation of
GS abundance. The functional consequence of a differentiated GS content between fiber types is unknown, since
we were unable to detect any differences in basal and
insulin-stimulated glycogen content in both fiber types.
This is likely due to the relatively small (,6%) increase in
glycogen content during a clamp procedure (46). If glycogen levels were solely dependent on GS, the activity of
this enzyme would be expected to be lower in type I
versus II fibers. However, our data cannot support this
because the higher expression and phosphorylation of GS
indicates that total GS activity is in fact higher in type I
versus II fibers. Thus, other factors than GS activity per se
determines glycogen levels.
In a recent study, Nellemann et al. (47) did not find
any changes in phosphorylation of PDH-E1a in human
skeletal muscle in response to insulin. Interestingly,
in the current study, PDH-E1a phosphorylation was decreased by insulin in type II fibers only. Thus, results by
Nellemann et al. (47) could have been influenced by
a muscle fiber type–dependent regulation not detected
in their whole-muscle biopsy preparation. An inverse relationship between PDH-E1a phosphorylation and PDHa
activity has been shown in human skeletal muscle during
exercise (48). Thus, findings in the current study suggest
an increased PDHa activity in response to insulin in type II
fibers only.
Study Limitations
All fiber pools were prepared from vastus lateralis muscle,
which expresses relatively small (,10%) amounts of type
IIx fibers (26). No significant differences in the MHC IIx
expression were observed between type II fiber pools
among the three groups (Supplementary Fig. 7). Thus,
differences between type I and II fiber pools observed in
the current study are likely not influenced by differences
in protein abundance/regulation between type IIa and IIx
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495
fibers. No measure of physical activity was performed. It
has been shown that training-induced increases in GLUT4
content mainly occur in type I fibers (22). Thus, training
status of the subjects in the current study could potentially influence differences between muscle fibers and/or
groups. All measures were performed in muscle fibers from
the vastus lateralis muscle. Whether fiber type–specific
differences in protein expression can be extended to other
muscles is unknown, but has been challenged by one
study (30), in which GLUT4 expression was higher in
type I versus IIa and IIx fibers from vastus lateralis
muscles but similar between fiber types in soleus and triceps brachii muscles. The current study design did not
allow exploration of this further. To evaluate the biological impact of fiber-specific signaling events further, the
methods used in the current study could be combined
with ex vivo incubation of human muscle strips (12)
and the recently described method of single-fiber glucose
uptake measurements (7). Such design demands open
surgical biopsies and was therefore not applicable to the
cohort of the current study.
In conclusion, based on protein level measures, the
enzymatic capacities for glucose uptake, phosphorylation,
and oxidation as well as for glycogen synthesis are higher
in human type I compared with type II muscle fibers. In
response to insulin, most differences in phosphorylation
between fiber types were due to differences in protein
levels. Thus, sensitivity for phosphoregulation by insulin
of these proteins is similar between fiber types. Even
though insulin-induced GDR was decreased in patients
with type 2 diabetes compared with lean and obese subjects,
few group differences in the muscle fiber–specific measurements were observed. However, our observations favor the
idea that fewer type I fibers and a higher number of type IIx
fibers in muscles from T2D patients contributes to the reduced GDR under insulin-stimulated conditions compared
with lean and obese subjects.
Acknowledgments. The authors thank M. Kleinert (University of Copenhagen, Denmark) for sharing know-how on the mTOR/NDRG1 analyses. The
authors also thank the following for the donation of material essential for this
work: L.J. Goodyear (Joslin Diabetes Center and Harvard Medical School,
Boston, MA), O.B. Pedersen (University of Copenhagen, Denmark), and J. Hastie
and D.G. Hardie (University of Dundee, U.K.). The monoclonal antibodies against
MHC I and II isoforms (A4.840 and A4.74) were developed by H.M. Blau, and
antibody directed against MHC IIx (6H1) was developed by C. Lucas. All MHC
antibodies were obtained from the Developmental Studies Hybridoma Bank
developed under the auspices of the National Institute of Child Health and
Human Development and maintained by The University of Iowa, Department
of Biology, Iowa City, IA.
Funding. This work was carried out as a part of the research programs
“Physical activity and nutrition for improvement of health” funded by the University of Copenhagen Excellence Program for Interdisciplinary Research and
the UNIK project Food, Fitness & Pharma for Health and Disease (see www
.foodfitnesspharma.ku.dk) supported by the Danish Ministry of Science, Technology and Innovation. This study was funded by the Danish Council for Independent Research Medical Sciences, the Novo Nordisk Foundation, and a Clinical
Research Grant from the European Foundation for the Study of Diabetes.
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Muscle Fiber Types and Insulin Signaling
Duality of Interest. P.H.A. and J.N. are employees at Novo Nordisk A/S
and own stocks in Novo Nordisk A/S. No other potential conflicts of interest
relevant to this article were reported.
Author Contributions. P.H.A. was responsible for conception and design
of research, performed analysis, interpreted results, drafted the manuscript,
edited and revised the manuscript, and approved the final version. A.J.T.P.
performed in vivo experiments and analysis, edited and revised the manuscript,
and approved the final version. J.B.B. performed analysis, interpreted results,
edited and revised the manuscript, and approved the final version. D.E.K. performed analysis, edited and revised the manuscript, and approved the final
version. B.F.V. performed in vivo experiments and analysis, edited and revised
the manuscript, and approved the final version. O.B. and J.N. edited and revised
the manuscript and approved the final version. K.H. interpreted results, edited
and revised the manuscript, and approved the final version. J.F.P.W. was responsible for conception and design of research, interpreted results, drafted the
manuscript, edited and revised the manuscript, and approved the final version.
J.F.P.W. is the guarantor of this work and, as such, had full access to all the data
in the study and takes responsibility for the integrity of the data and the accuracy
of the data analysis.
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